PROJECT SUMMARY The LonA protease, which belongs to the superfamily of ATPases associated with diverse cellular activities (AAA+ ATPases), is a proteolytic enzyme responsible for protein quality control and the post-translational regulation of a wide array of cellular processes. In Vibrio cholerae, the LonA controls cellular processes (i.e., biofilm formation, motility, Type-6 secretion system, and intestinal colonization) that are critical for the pathogen's infection cycle. While we know that LonA is a key regulator in V. cholerae, the molecular mechanisms by which LonA controls these cellular processes remains unclear. The experiments in this proposal will fill key gaps in our understanding of the mechanisms of regulated proteolysis by the LonA protease. We hypothesize that a key signaling molecule, cyclic dimeric guanosine monophosphate (c-di-GMP), modulates LonA activity and/or substrate selectivity. In the first aim, we will build on our observations that c-di-GMP modulates LonA activity and that TfoY is a LonA target. We will utilize biochemical and genetic approaches to determine the in vivo and in vitro consequences of c-di-GMP binding to LonA using reporter substrates. In the second aim, we will use proteomic approaches to determine the c-di-GMP dependent substrate profiles of LonA in biofilms where c-di- GMP levels are high, and in mutants with raised or lowered cellular c-di-GMP levels. We will determine in vivo and in vitro stability of selected substrates in wild-type, Lon-deficient and LonAcdGMP-null strains. This proposal takes advantage of the Yildiz lab's expertise in c-di-GMP signaling, biofilm formation and V. cholerae biology and the Chien lab's strength in biochemical reconstitution of AAA+ proteases to determine the role of c-di-GMP on the Lon family of proteases. Completion of our studies will provide a mechanistic understanding of the broadly conserved post-translational regulator, Lon, and the signaling molecule, c-di-GMP, as well as identify targets that can be used to inhibit cellular processing contributing to virulence of bacterial pathogens.